A large diverse group of nonmotile, heterotrophic, eukaryotic organisms are collectively referred to as fungi. Most fungi are saprophytes, i.e. securing their food from dead organic material. Because of their heterotrophic properties, i.e. using organic material as carbon source, many fungi produce metabolites of industrial interest. Certain species are further useful as sources for food, whereas others are responsible for spoiling almost any organic material in which they come in contact.
Fungal cells range in size from microscopic unicellular organisms to macroscopic as e.g. mushrooms. True fungi are in general terrestrial and includes Zygomycetes, such as Rhizopus, Basidiomycetes such as Puccinia graminis, Ascomycetes, such as Neurospora and Saccharomyces and Deuteromycetes, such as Penicillium and Aspergillus.
Fungal cells
Fungal microorganisms include multicellular as well as unicellular organisms and are in general considered to consist of yeasts and molds. Unicellular fungi are primarily yeasts, while the term mold is used for fungi that are predominantly mycelial.
Fungal cells have a quite complex structure, constituting of a cytoplasm, comprising nucleus, mitochondria, microbodies etc. encapsulated by a cytoplasmic membrane. Chemically and structurally the cytoplasmic membrane consist of a bilayer of phospholipids with different proteins inserted into it.
The cytoplasmic membrane is surrounded by the rigid cell wall.
Structure of fungal cell walls
The cell walls of most true fungal microorganisms contain a network of glucan, which gives the cell wall strength. Further major fungal cell walls constituents are mannoprotein and chitin.
Glucan and chitin is far more resistant to microbial degradation than cellulose, which is the major constituent of the cell wall of many fungi-like organisms, such as Oomycetes.
Glucan is predominantly .beta.-1,3-linked with some branching via 1,6-linkage (Manners et al., Biotechnol. Bioeng, 38, p. 977, 1973), and is known to be degradable by certain .beta.-1,3-glucanase systems.
.beta.-1,3-glucanase includes the group of endo-.beta.-1,3-glucanases also called laminarinases (E.C. 3.2.1.39 and B.C. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc, 1992).
Pegg et al., Physiol. Plant Pathol., 21, p. 389-409, 1982, showed that a purified endo-.beta.-1,3-glucanase from tomato in combination with an exo-.beta.-1,3-glucanase of fungal origin were capable of hydrolysing isolated cell wall of the fungus Verticillium alboatrum.
Further, Keen et al., Plant Physiol., 71, p. 460-465 showed that a purified .beta.-1,3-glucanase from soy bean was capable of degrading isolated cell walls of fungi.
Large scale degradation of the fungal cell wall
The unit operation of cell disruption appears as an essential first step for intracellular products separation and downstream processing of valuable intracellular products.
Large scale cell disruption is in general carried out by rather vigorous treatment involving the use of strong chemicals and/or mechanical means. This leaves the target protein with a very complex mixture of contaminants.
In this context extensive industrial implementation of alternative approaches to conventional microbial cell disruption techniques are becoming of increasing relevance (Asenjo et al., Bio/technol, 11, p. 214, 1993; De la Fuente et al., Appl. Microbiol. Biotechnol, 38, p. 763, 1993).
Selective Cell Permeabilization (SCP) and Selective Protein Recovery (SPR) as a mean to increase bio-process productivity, economy and product quality by simplifying the downstream processing of intracellular products have proved to be very attractive in terms of their delicacy and specificity (Asenjo et al., 1993, supra; Shen et al., Gene, 84, P. 1989).
SCP and SPR involves the use of pure preparations of cell-wall-degrading .beta.-glucanases to increase fungal cell wall porosity (with very limited cell lysis) and facilitate the release of intracellular proteins. In this way, SCP gives primary separation of the target product from some of its major contaminants. A major limitation to this approach is the relatively low level of expression of yeast lytic enzymes presently obtained in the bacteria used for the production of these enzymes (e.g. Oerskovia xanthineolytica, Andrews and Asenjo, Biotech. Bioeng, 30, p. 628, 1987). Today Oerskovia xanthineolytica are sometimes referred to as Cellulomonas cellulans. However, the name Oerskovia xanthineolytica will be used below.
A number of commercial enzyme compositions useful in the enzymatic lysis of fungal cells are available. Such products normally comprise multiple enzymatic activities, e.g. including .beta.-1,3- and .beta.-1,6-glucanase, protease, chitinase, mannase and other enzymes capable of degrading cell wall components.
The lytic system of Oerskovia xanthineolytica LLG109
The lytic enzyme system of Oerskovia xanthineolytica LLG109 has partially been isolated and purified and some of the glucanase and protease components have been characterised (Ventom and Asenjo, Enzyme Microb. Technol., 13, p. 71, 1991).
Although a single molecular species of lytic .beta.-1,3-glucanase has been characterized from O. xanthineolytica LLG109, most Oerskovia strains seem to have multiple .beta.-1,3-glucanase systems (Doi and Doi, J. Bacteriol., 168, p. 1272, 1986).
While all observed molecular forms of these enzymes possess hydrolytic activity towards .beta.-1,3-glucans (.beta.-1,3-glucanase activity), only some are found capable of readily solubilizing yeast glucan and inducing lysis of viable yeast cells.
In contrast, other types of endo-.beta.-1,3-glucanases would solubilize yeast glucan only partially, causing only limited cell lysis (Doi and Doi, supra, 1986). However, this multitude of enzyme species produced by Oerskovia may be partially due to proteolytic processing.
The genetic relationship between these enzymes is still unclear, as the number of yeast lytic enzymes so far cloned is very limited. As a result, there is still limited knowledge about the gene structure and protein function relationship (Shen et al, J. Biol. Chem, 266, p. 1058, 1991; Shimol and Tademura, J. Biochem, 110, 608, 1991; Watanabe et al., J. Bacteriol, 174(1), p. 186, 1992; Yamamoto et al., Biosci. Biotechnol. Biochem., 57, p. 1518-1525, 1993).
Characterization of yeast lytic enzymes from O. xanthineolytica
A purified lytic .beta.-1,3-glucanase showed a molecular mass of about 31 kDa, when estimated by SDS-PAGE and a pI of 5.0. The amino acid composition was also determined. The yield was optimized by the continuous culture process, but yields were still low.
The specific activity of the enzyme was 11.1 U/mg. The K.sub.m for .beta.-1,3-glucanase activity on yeast glucan was 2.5 mg/ml for laminarin (a soluble .beta.-1,3-glucan) 0.95 mg/ml. The pH optimum for .beta.-1,3-glucanase was 8.0 on yeast glucan and 6.0 on laminarin substrate. The lytic .beta.-1,3-glucanase caused only limited lytic activity on viable yeast (Saccharomyces cerevisiae) cells (Ventom and Asenjo, supra, 1991), but this was stimulated synergistically by the lytic protease component.
In addition, there was detected another .beta.-1,3-glucanase component in clarified O. xanthineolytica continuous fermentation broth, although it was not purified to homogeneity and subsequently characterized.
Patent documents
A number of .beta.-1,3-glucanase genes and uses thereof have been sought patented.
An example is DD 226012 (Akad. Wissenshaft DDR) which concerns a method for production of a Bacillus .beta.-1,3-glucanase. Further, JP 61040792 A (DOE K) describes a cell wall-cytolase .beta.-1,3-glucanase recombinant plasmid for removing the cell walls of yeast. The gene is derived from Arthrobacter and is transformed into Escherichia group bacteria.
EP 440.304 concerns plants provided with improved resistance against pathogenic fungi transformed with at least one gene encoding an intracellular chitinase, or an intra- or extracellular .beta.-1,3-glucanase. The matching recombinant polynucleotides is also disclosed.
WO 87/01388 describes a method for preparing cell lytic enzymes, such as .beta.-1,3-glucanases, which can be produced by Oerskovia.
WO 92/03557 discloses a recombinant DNA expression vector comprising a 2.7 kb DNA sequence, derived from Oerskovia xanthineolytica, encoding a .beta.-1,3-glucanase. Said glucanase, expressed in E. coli, exhibits glucanase activity and no protease activity. E. coli has a number of deficiencies in connection with large scale industrial enzyme production. First of all the glucanase is expressed intercellular. Consequently the cells need to be opened to get access to the enzyme. This makes recovery of the enzyme cumbersome and expensive.
From WO 92/16632 a recombinant DNA sequence coding for a novel protein with .beta.-1,3-glucanase activity is known. The gene is derived from soy.
Most presently available enzyme preparations for the use of lysing furngal cells contain protease activity, which leaves the lysed target protein with a very complex mixture of contaminants.
It is therefore desirable to provide a .beta.-1,3-glucanase substantially free of protease activity which is capable of opening the cell walls in a gentle way. This will facilitate the recovery and purification of the target protein.
Further, it would be advantageous to express the gene encoding the target protein in a heterologous host cell capable of increasing the production yield.